The RecA protein of E. coli, which is both a recombinease and a so-called protease, has a central role in repair of damaged DNA; both activities participate in repair. The protease function of the RecA protein involves at least three functional sites, namely a binding site for the substrate whose cleavage is thereby enhanced, and two effector-binding sites whose states determine whether or not the binding site is active. One aim of this project is to determine the location, structure, and interaction of the three sites of RecA protease function and to determine how they are related to the sites for recombinase function. A mutational approach is being used. Mutations called recA (Prt-c) confer constitutive protease activity on RecA protein without the usual need for DNA damaging agents. In vitro studies of the effector- and substrate- binding properties of various recA (Prt-c Rec+) and recA (Prt-c Rec-) mutants will be carried out to determine where on the RecA polypeptide the active sites are located and how their functional coupling is accomplished. New recA mutants, including suppressors and double mutants, will be isolated or constructed and their genes sequenced, which will also eanble one to determie which reigons of the RecA polypeptide are functionally coupled. A sequence analysis of new recA mutants that alter substrate recognition will identify the substrate binding site and aid in explaining how the binding is coupled functionally to the effector- binding sites. The differences between the direct role of the RecA protein in spontaneous and UV-induced mutagenesis and its indirect role in enhancing proteolyis of repressors will be studied and the role of putative cryptic lesions in spontaneous mutation will be tested. These experiments are a continuation of our general aim to provide a framework for a definitive understanding of the function of the RecA protein.